Name: scanning electron microscopy (sem) protocols for problematic plant, oomycete, and fungal samples
Uploaded: 2017-02-03T13:00:00
Description: Watch this Scientific Journal Video about Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples at JoVE.com

This project has received funding from the European Union&#39;s Horizon 2020 research and innovation programme under grant agreement No. 634429. This publication reflects the views only of the author, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. We also acknowledge the financial contribution made by the Real Jard&#237;n Bot&#225;nico, CSIC. SR is grateful to the European Union [ITN-SAPRO-238550] for the support of her research in Saprolegnia. We also want to thank Francisco Calonge for kindly provide the Phellorinia herculanea images and B. Pueyo for processing samples (Figure 5). All images were taken by the SEM service at the Real Jard&#237;n Bot&#225;nico-CSIC in Madrid.

NOTE: This protocol includes six main sections, three devoted to specific organisms (sections 1-3), and three describing the procedures common to all (4-6). Asterisks (*) indicate steps modified by the experimenters.
1. Studies of Developing and Fully Formed Plant Structures
<ol>
	<li>Collection and fixation
	<ol>
		<li>If the plant material is collected in a place with no access to a fume cupboard, introduce and immerse the material in 70% ethanol in centrifuge tubes. Ideal...

With respect to standard SEM protocols, the procedures presented here include relatively rapid, easy to follow, and low-cost methodologies. Depending on the amount of samples and on the ease of processing, it takes four to five days to acquire good quality images. Including adequate safety precautions for the CPD and SEM operation, the procedures are easy to handle. Particular caution should be taken with formalin and the glutaraldehyde (see steps 1.1.1 to 1.1.3 and 2.1.5 of the protocol). There are certain steps where, ...

In biology, the use of scanning electron microscopy (SEM) has been extended to studies of structural evolution, comparative morphology, organ development, and characterization of populations or species1. With its two-dimensional view of microscopic structures, areas such as micromorphology and systematics profited from SEM technique advances since the second half of the 20th century. For example, the introduction of the sputter coating methodology in the 1970s made possible observations of delicate materials such as shoot apices and flowers enhancing the imaging of non-conductive tissues2,3. SEM uses electrons ejected from the surface of the specimen to reproduce the topography in a high-vacuum environment4.
Studies involving SEM are focused in both the inference of structural characters and the reconstruction of growth processes. New structural characters relevant to the taxonomy and systematics of a wide range of organisms have been discovered from SEM observations. For example, plant traits used for species diagnosis or supraspecific classifications, such as the vestured pits of wood5, stigma diversity6, nectary and floral morphology7,8, trichome details9, and pollen grains10,11, cannot be properly visualized without SEM. Successful observations with conventional SEM have been also achieved for long-time formalin-fixed organisms12 and plant herbarium specimens13.
On the other hand, studies of reconstruction of growth processes using SEM involve a wide range of topics, such as organ development14, infections induced by bacteria15, plant root physiology16, parasite-host attachment mechanisms17,18, drug effects on parasites19, mycoparasitism and antibiosis20,21, growth malformation22, comparative development of wild and mutant individuals23, and entire life cycles24. Although environmental scanning electron microscopes (ESEM)25 may have important advantages for the observation of wet biological samples in growth processes, delicate material may still be compromised even in the low vacuum condition of the ESEM), and need to be processed adequately to avoid loss of valuable morphological observation.
In this paper, a review of specific protocols for SEM observation of three different types of samples is presented: floral meristems, oomycetes (Saprolegnia), and fungal material. These protocols compile the experience of our previous SEM-based studies26,27,28,29,30,31,32,33, where specific difficulties and alternative solutions have been found. In the case of plant comparative developmental and structural studies, the use of SEM started in the 1970s34,35, and since then, researchers discovered that certain floral features are more labile than previously thought36. Reconstruction of floral development involves the capture of all stages between young floral meristems and anthesis. To reach this aim, it is essential that the sample topography and the cell wall integrity are not compromised after the fixation and subsequent dehydration. Young floral meristems are particularly vulnerable to cell wall collapse (Figures 1a, 1b). Similarly, delicate structures such as nectaries, petals, stigmas and sporangia require effective and undamaging protocols. This review summarizes an optimal protocol to keep young and delicate tissues intact for SEM imaging.
In the case of the oomycetes (Stramenopiles)-one of the most diverse and widespread groups of parasites, with hosts ranging from microbes and plants to invertebrates and vertebrates37- there are spores that grow and develop in a wet environment. This condition represents a challenge for SEM observation because the spores need an adequate substrate not suitable for standard SEM protocols. Among the oomycetes, species of Saprolegnia are of particular interest because they can cause severe reductions in aquacultures, fisheries, and amphibian populations38. Micromorphological characteristics, such as the hooked spines of cysts, have been found to be useful to identify species of Saprolegnia, which is fundamental to establish infection controls and potential treatments39. Here, there is an experimental protocol to compare the patterns of the spine growth of cysts on different substrates and to manipulate the sample for critical point dryer (CPD) preparation and subsequent SEM observation.
In a third case, there are interesting findings that came up after an inspection of the spores of the fungi Phellorinia herculanea f. stellata f. nova (Agaricales)31. Together with the spores, a group of unexpected nursery cells was identified under the SEM. With previous traditional protocols and untreated material, the nurse cells came out completely collapsed (Figure 1c). Further inferences about particular tissues associated to the spores can be made with the simple but crucial modifications to the standard approaches described here (Figure 1d).
In this review, there are detailed SEM protocols that can be used to deal with different problems associated with SEM observation in angiosperms, oomycetes, and Agaricales, such as cell collapse and meristematic tissue shrinking, non-optimal growth of cyst spines, and destruction of ephemeral tissues, respectively.
<img alt="Figure 1" src="/files/ftp_upload/55031/55031fig1.jpg" /> 
Figure 1: Comparison of samples treated without (a, c) and with (b, d) the protocol FAA-ethanol-CPD. (a-b) Floral buds of Anacyclus clavatus, mid-development. Bud treated with osmium tetroxide46 (a) and bud treated with the FAA-CPD protocol (b). (c-d) Nurse cells with spores of Phellorinia herculanea f. stellata. Dried samples without any treatment (c) and with the protocol here described for Agaricales (d). Spores in orange. Scales: (a-b) 100 &#181;m, (c-d) 50 &#181;m. Photos were taken by Y. Ruiz-Le&#243;n. <a href="http://cloudflare.jove.com/files/ftp_upload/55031/55031fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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scanning electron microscopy (sem) protocols for problematic plant, oomycete, and fungal samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Floral Development and Fixation of Developing and Fully Formed Plant Structures
Using the FAA-CPD protocol described here, young and mature plant tissues are optimally fixed and dehydrated for SEM imaging. Processes such as floral development can be reconstructed because the topography and shape of the buds is not distorted by cell shrinking (Figures 1b, 1d, 4a-f). Structures with complex shapes can be successfull...

Watch this Scientific Journal Video about Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples at JoVE.com

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

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